The present invention is an apparatus for separating unknown solutes in a buffer solution. This invention is particularly concerned with the recognition of large biological materials which are transported through a fine capillary using chemical electrophoresis techniques.
Recent advances in biotechnology have accelerated the demand of research laboratories, health-care facilities, and pharmaceutical manufacturers for devices which are capable of accurately and rapidly identifying extremely small quantities of inorganic and organic substances. Previous techniques, which include gas and liquid chromatography, have been employed to assay samples whose molecular structure is relatively small. Although chromatography may be successfully employed to resolve mixtures of inorganic and small organic molecules; the very large and exceedingly complex molecules of amino acids, proteins, peptides, and DNA are more difficult to separate than the smaller molecules. Another serious flaw in chromatographic methods is the predicament that is encountered when the unknown sample is especially sparse, since chromatography utilizes relatively large amounts of the material which requires analysis. Other shortcomings that are experienced when liquid chromatography is practiced include inconsistent patterns of solute movement through the system which creates uneven flows called "dead zones" and undesirable laminar mixing as opposed to plug movement of the fluids.
Electrophoresis is another well-known procedure that enables the researcher or scientist to evaluate undetermined materials. Capillary electrophoresis utilizes an electric field which is imposed across a length of tubing or capillary that contains a mixture of the unknown sample and a non-reactive liquid often called a buffer solution. The electric field creates an electro-osmotic flow and causes the buffer and the constituents of the unknown sample to be pulled through the capillary. The electric field also creates a superimposed electrophoretic flow which separates the constituents of the sample according to their varying molecular drags and varying net electrical charges. Because dissimilar substances do not react to the drag and electrical attraction in the same way, they become increasingly separated into distinct zones or groups as they progress along the capillary. Each band of constituent material that makes up the original unseparated mixture of unknown material passes through the capillary by itself. At some point along this tubing, each band is examined and identified by a detector. One type of detector for electrophoretic separations measures the electrical conductivity of the bands in the capillary. An alternative detection scheme is a method called laser induced fluorescence. Although this technique is highly sensitive, it is costly and is limited to detecting compounds that fluoresce or which can be stimulated to do so.
Another previous system probes the unknown material by shining ultra-violet light through an unknown solute borne by a buffer solution and then gauging how much light is absorbed by the solute. This radiation absorption method is the most popular technique among the various available electrophoretic systems. The capillaries in this system must be kept very narrow so that all the material inside it moves without turbulence or eddy currents. This uneven flow is created when the electrical current that passes through the contents of the capillary heats the buffer solution. This heat is conducted radially from the center of the tube out to its periphery. The migration of heat establishes a radial thermal gradient, extending from a relatively hot zone at the center of the capillary to cooler temperatures at the capillary wall. These different heat levels across the vessel set up convection currents within the buffer solution that tend to mix the contents of the capillary in directions that are perpendicular to the flow of liquid and solute through the capillary tube. This mixing destroys the sensitivity of the electrophoretic separation instrument because it tends to broaden the bands of separated solute. Another problem caused by this uneven heating in the capillary is the creation of viscosity gradients that also tend to broaden the solute bands. Most separation systems are also beset by an undesirable condition called gravity siphoning. When the two ends of the separation capillary are not precisely aligned, the difference in their heights causes an unwanted flow due to the efforts of gravitational forces to equalize the height of the contents of each column.
Conventional systems have been designed to minimize unwanted turbulence, viscosity gradients, and gravity siphoning by utilizing capillaries that are extremely narrow. Capillaries having relatively small cross-sections reduce uneven heating which generates unwanted eddy currents and viscosity gradients. The detriment suffered by this compromise is the severe reduction in sample size that can be analyzed by the detector at any given time. This relatively small sample volume which may be processed at any one time is a serious difficulty that is shared by all previous detection methods. To prevent undesirable dispersion of the solute caused by large thermal gradients, the diameter of conventional circular capillaries is usually kept at or below one hundred microns. Fifty microns is a common size for these capillaries. As a consequence, the sensitivity of conventional systems is limited to a level far below the one-part-per-million threshold, which is the current desirable detection accuracy which should be achieved to analyze mixtures of inorganic and small organic molecules.
As the technology of genetic engineering continues to evolve, diagnostic and measurement techniques which are more accurate, reliable, and sensitive become increasingly more valuable. Doctors, clinicians, and laboratory technicians need more powerful tools to explore the intricacies of the genetic code, to improve the hardiness and usefulness of plant and animal life in an effort to feed the world's burgeoning population, and ultimately to devise cures for inherited disabilities and dreaded diseases. The problem of providing a highly sensitive and precise separation system that overcomes the limitations that impair previous devices and techniques has presented a challenge to designers and innovators in the biochemical arts. The development of an effective and sensitive system for separating the components of an unknown biological sample would constitute a major technological advance in the biochemical and biotechnology industries. The enhanced performance that could be achieved using such an innovative device would satisfy a long felt need within the business and would enable manufacturers of drugs, medicines, and biological products to save substantial expenditures of time and money.